Cytokine-Like Proteins

ABSTRACT

A full-length cDNA corresponding to an EST (AA418955), which does not show any homology to other proteins in the database but has a weak homology to G-CSF, has been successfully isolated by synthesizing primers based on the EST sequence, and effecting PCR-cloning from a human fetal spleen library. Sequencing of the thus-isolated cDNA and analysis of its structure revealed that the cDNA has typical characteristics of a factor belonging to the IL-6/G-CSF/MGF family. It is also found out that the culture supernatant of said sequence-transfected CHO cells shows a proliferation supporting activity towards bone marrow cells in the coexistence of kit ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.11/716,808, filed on Mar. 12, 2007, which is a divisional of U.S.application Ser. No. 10/440,066, filed May 15, 2003, issued as U.S. Pat.No. 7,252,967, which is a divisional application of U.S. applicationSer. No. 09/687,637, filed Oct. 13, 2000, issued as U.S. Pat. No.6,610,285, which is a continuation-in-part of International PatentApplication No. PCT/JP99/01997, filed Apr. 14, 1999, and claims priorityfrom Japanese Patent Application No. 10-121805, filed Apr. 14, 1998. Thedisclosures of the foregoing applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention relates to a novel cytokine-like protein and the encodinggene.

BACKGROUND OF THE INVENTION

Cytokines are multi-functional cell growth/differentiation inducingfactors controlling immune and hematopoietic reactions. The series offactors composing cytokines, are mainly produced by activated T cells,macrophages, or stromal cells and connect the cells of the lymphoidsystem and the hematopoietic system in a network, regulating theproliferation, differentiation, and functions of these cells. So far, anumber of factors have been isolated as cytokines and apart from thefactors themselves, antibodies and receptor molecules of those factors,or antibodies against those receptors, have been developed astherapeutic drugs and are in actual use.

For example, G-CSF, which has a neutrophil-proliferating function, isalready in use as a drug for many diseases and leukopenia resulting fromthe treatment of these diseases (K. Welte et al., the first 10 yearsBlood Sep. 15, 1996; 88:1907-1929 and also refer GENDAIKAGAKU ZOUKAN no.18, Cytokine, edited by TOSHIAKI OHSAWA, 1990, published by TOKYO KAGAKUDOUJIN). Furthermore, an antibody against the receptor of IL-6, whichacts in immune functions and inflammation, is being developed as apotential therapeutic drug against rheumatism and leukemia.

SUMMARY OF THE INVENTION

The present invention provides a novel cytokine-like protein and theencoding DNA. It also provides a vector into which the DNA has beeninserted, a transformant carrying the DNA, and also a method forproducing a recombinant protein using the transformant. Furthermore,screening methods for a compound, which binds to the protein andregulates the activity, and the uses of the protein and the compoundsregulating its function as pharmaceutical drugs, are also provided bythe present invention.

Most cytokines known so far, have a conserved characteristic such as aWS Motif (Idzerda, R L et al., J Exp Med 1990 Mar. 1; 171 (3) 861-873),and form a super-family of cytokine receptors. Although the cytokineitself, which is the ligand, does not have a conserved characteristic orhomology compared to the receptor, some groups have an extremely weakhomology, hinting of a close stereoscopic structure. EPO/TPO family andIL-6/G-CSF/MGF family can be taken as examples. The present inventors,thinking that unknown, yet unidentified genes may exist in thesefamilies, attempted to isolate an unknown cytokine belonging to thesefamilies.

Specifically, it was found that the EST (AA418955) sequence, which doesnot show any homology to other proteins in the Database, has a weakhomology to G-SCF and constructed primers based on that sequence, anddid PCR cloning from a human fetal spleen library. As a result, thepresent inventors succeeded in isolating a full-length cDNAcorresponding to the EST (this clone was named SGRF). Also, the isolatedSGRF cDNA was sequenced and the structure analyzed to find that theisolated cDNA has the typical characteristics of a factor belonging tothe IL-6/G-CSF/MGF family. Furthermore, the inventors analyzed theactivity of the SGRF protein to find that the culture supernatant ofSGRF-transfected CHO cells has a proliferation supporting activitytowards specific bone marrow cells in the presence of mouse kit ligand.The isolated SGRF protein itself may be applicable for the preventionand treatment of diseases of the lymphoid and hematopoietic systems andfor diseases related to defective cell growth. Also it is possible touse this protein for the screening of other factors related to thelymphoid and hematopoietic systems and as a drug candidate compound fordiseases of those systems.

Namely, this invention relates to a novel cytokine-like protein SGRF andthe encoding gene, their production as well as the use of the protein inthe screening of drugs and drug candidate compounds. More specifically,

1. a protein comprising the amino acid sequence of SEQ ID NO:1, or saidsequence in which one or more amino acids are replaced, deleted, added,and/or inserted,

2. a protein encoded by a DNA hybridizing with the DNA comprising thenucleotide sequence of SEQ ID NO:2, which is functionally equivalent tothe protein having the amino acid sequence of SEQ ID NO:1,

3. a DNA encoding the protein of (1) or (2),

4. the DNA of (3), which contains the coding region of the nucleotidesequence of SEQ ID NO:2,

5. a vector in which the DNA of (3) or (4) is inserted,

6. a transformant carrying, in an expressible manner, the DNA of (3) or(4),

7. a method for producing the protein of (1) or (2), which comprises theculturing of the transformant of (6),

8. a method for screening a compound which can bind to the protein of(1) or (2), the method comprising the steps of:

(a) exposing a test sample to the protein of (1) or (2) or its partialpeptide;

(b) detecting the binding activity between the test compound and saidprotein or its partial peptide; and

(c) selecting a compound having a binding activity to said protein,

9. a compound which can bind to the protein of (1) or (2),

10. the compound of (9) which is obtainable by the method of (8),

11. a method for screening a compound which can promote or inhibitactivity of the protein of (1) or (2), the method comprising the stepsof:

(a) exposing the protein of (1) or (2) and the kit ligand to mammalianbone marrow cells under the absence of a test compound;

(b) detecting the proliferation of said bone marrow cells; and

(c) selecting a compound which promotes or inhibits the proliferation ofbone marrow cells in comparison with the assay under the presence of thetest sample,

12. the method of (11), wherein the bone marrow cells are Lin negative,Sca-1 positive, c-kit positive, and CD34 positive,

13. a compound which promotes or inhibits the activity of the protein of(1) or (2),

14. the compound of (13) which is obtainable by the method of (11) or(12),

15. a pharmaceutical composition comprising the protein of (1) or (2) asan active component,

16. a promoter or inhibitor of the protein of (1) or (2) wherein theactive component is the compound of (13) or (14),

17. an antibody which can bind to the protein of (1) or (2), and

18. a DNA comprising at least 15 nucleotides, which can specificallyhybridize with the DNA comprising the nucleotide sequence of SEQ IDNO:2.

The present invention relates to a novel cytokine-like protein. Thenucleotide sequence of the cDNA encoding the protein named “SGRF”, whichis included in the protein of the present invention is shown in SEQ IDNO: 2; the amino-acid sequence of said protein in SEQ ID NO:1.

So far, in mammals, IL-6 and G-CSF have been reported as factors thoughtto belong to the IL-6/G-CSF/MGF family. The “SGRF” cDNA isolated by thepresent inventors, had in its 3′ non-coding region, four mRNAdestabilizing sequences (Lagnando C A, Brown C Y, Goodall G J (1994)Mol. Cell. Biol. 14, 7984-7995) called ARE (AT Rich element), often seenin cytokine mRNAs. The consensus sequence preserved in theIL-6/G-CSF/MGF family was also roughly maintained (FIG. 3). From thesefacts, “SGRF” can be assumed to be a novel factor belonging to theIL-6/G-CSF/MGF family.

The “SGRF” expression in human normal tissue as detected bynorthern-blot analysis is extremely localized, and was seen in thetestis, lymph nodes, and thymus, being not present in a detectable levelin other tissues (FIG. 4). Even in tissues where expression wasdetected, the expression level was assumed to be very low. An EST(U38443), a partial fraction of “SGRF”, which is normally hardlyexpressed, is reportedly induced following activation in a T cell-line(Jurkat) (Yatindra Prashar, Sherman M. Weissman (1996) Proc. Natl. Acad.Sci. USA 93:659-663). From this fact and from the results ofnorthern-blot analysis, it can be assumed that in vivo, “SGRF” is mainlyexpressed in activated T cells.

Furthermore, the culture supernatant of “SGRF” transfected CHO cellsshowed an activity, which supported the proliferation of bone marrowcells (FIG. 12), in the presence of the kit ligand.

The characteristics of “SGRF” such as those above, suggest that it is akind of a typical interleukin. “SGRF”, as are most cytokines isolated sofar, is thought to be involved in the lymphoid and hematopoieticsystems. Therefore, it can be applied as a therapeutic or preventivedrug in diseases of the lymphoid and hematopoietic systems, and also indiseases associated with defects in cell proliferation.

The protein of the present invention can be prepared by methods commonlyknown to one skilled in the art, as a recombinant protein made usinggenetic engineering techniques, and also as a natural protein. Forexample, a recombinant protein can be prepared by, inserting DNAencoding the protein of the present invention (for example, DNAcomprising the nucleotide sequence in SEQ ID NO:1) into a suitableexpression vector, introducing this into a host cell, and purifying theprotein from the resulting transformant or the culture supernatant. Thenatural protein can be prepared by immobilizing in a column, antibodiestaken from immunizing a small animal with the recombinant protein, andperforming affinity chromatography for extracts of tissues or cells (forexample, testis, lymph nodes, thymus, etc.) expressing the protein ofthe present invention.

Also, this invention features a protein, which is functionallyequivalent to the “SGRF” protein (SEQ ID NO:1). The method of insertinga mutation into the amino acids of a protein is a well-known method forisolating such proteins. In other words, for a person skilled in theart, the preparation of a protein functionally equivalent to the “SGRF”protein, is something which can be generally done using various methodssuch as the PCR-mediated, site-specific-mutation-induction system(GIBCO-BRL, Gaithersburg, Md.), oligonucleotide-mediated,site-specific-mutation-induction method (Kramer, W. and Fritz, HJ (1987)Methods in Enzymol., 154:350-367) suitably replacing amino acids in the“SGRF” protein shown in SEQ ID NO:1, which do not influence thefunction. Mutations of amino acids can occur spontaneously as well.Therefore, the protein of the invention includes those proteins that arefunctionally equivalent to the “SGRF” protein, having an amino acidsequence in which one or more amino acids in the amino acid sequence ofthe “SGRF” protein (SEQ ID NO:1) have been replaced, deleted, added,and/or inserted. The term “functionally equivalent” as used herein,refers to a protein having a cytokine activity equivalent to that of the“SGRF” protein. The cytokine activity of the “SGRF” protein includes,for example, a proliferation-supporting activity (Example 11) towardscells which are Lin negative, Sca-1 positive and c-kit positive.

The number of amino acids that are mutated is not particularlyrestricted, as long as a cytokine activity equivalent to that of the“SGRF” protein is maintained. Normally, it is within 50 amino acids,preferably within 30 amino acids, more preferably within 10 amino acidsand even more preferably within 5 amino acids. The site of mutation maybe any site, as long as a cytokine activity equivalent to that of the“SGRF” protein is maintained.

A “conservative amino acid substitution” is one in which an amino acidresidue is replaced with another residue having a chemically similarside chain. Families of amino acid residues having similar side chainshave been defined in the art. These families include amino acids withbasic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

In the present invention, the protein having numerous depletions in theamino acid sequence of the “SGRF” protein (SEQ ID NO:1) includes apartial peptide. The partial peptide includes, for example, a protein ofwhich the signal peptide has been excluded from the “SGRF” protein ofSEQ ID NO:1.

Also, a fusion protein can be given as a protein comprising the aminoacid sequence of the “SGRF” protein and several amino acids addedthereto. Fusion proteins are, for example, fusions of the abovedescribed proteins and other peptides or proteins, and are included inthe present invention. Fusion proteins can be made by techniquescommonly known to a person skilled in the art, such as linking the DNAencoding the protein of the invention and with DNA encoding otherpeptides or proteins, so as the frames match, inserting this into anexpression vector and expressing it in a host. There is no restrictionas to the peptides or proteins fused to the protein of the presentinvention.

Commonly known peptides, for example, FLAG (Hopp, T. P. et al.,Biotechnology (1988) 6:1204-1210), 6×His constituting six His(histidine) residues, 10×His, Influenza agglutinin (HA), human c-mycfragment, VSP-GP fragment, p18HIV fragment, T7-tag, HSV-tag, E-tag,SV40T antigen fragment, lck tag, α-tubulin fragment, B-tag, Protein Cfragment can be used as peptides that are fused to the protein of thepresent invention. Examples of proteins that are fused to protein of theinvention are, GST (glutathione-S-transferase), HA (Influenzaagglutinin), Immunoglobulin constant region, β-galactosidase and MBP(maltose-binding protein). Fusion proteins can be prepared by fusingcommercially available DNA encoding these peptides or proteins with theDNA encoding the protein of the present invention and expressing thefused DNA prepared.

The hybridization technique (Sambrook, J. et al., Molecular Cloning 2nded. 9.47-9.58, Cold Spring Harbor Lab. press, 1989) is well known to oneskilled in the art as an alternative method for isolating a proteinfunctionally equivalent to the “SGRF” protein (SEQ ID NO:1). In otherwords, for a person skilled in the art, it is a general procedure toprepare a protein functionally equivalent to the “SGRF” protein, byisolating DNA having a high homology with the whole or part of the DNAencoding the “SGRF” protein used as a base for the preparation of theprotein. Therefore, the protein of the present invention also includesproteins, which are functionally equivalent to the “SGRF” protein andare encoded by DNA hybridizing with DNA encoding the “SGRF” protein. Theterm “functionally equivalent” as used herein, means as mentioned above,proteins that show a cytokine activity equivalent to that of the “SGRF”protein. Apart from humans, for example, mice, rats, cows, monkeys andpigs can be used as animals from which functionally equivalent proteinscan be isolated. One skilled in the art can suitably select thestringency of hybridization for isolating DNA encoding a functionallyequivalent protein, but normally, it is equilibrium hybridization atabout 42° C., 2×SSC, 0.1% SDS (low stringency); about 50° C., 2×SSC,0.1% SDS (medium stringency); or about 65° C., 2×SSC, 0.1% SDS (highstringency). If washings are required to reach equilibrium, then thewashings are performed using the same buffer as the originalhybridization solution, a listed above. In general, the higher thetemperature, the higher is the homology of the DNA obtainable. “Highhomology” refers to, in comparison with the amino acid sequences of the“SGRF” protein, normally a homology of 40% or higher, preferably 60% orhigher, more preferably 80% or higher, even more preferably 95% orhigher. The homology of a protein can be determined by following thealgorithm in Wilbur, W. J. and Lipman, D. J. Proc. Natl. Acad. Sci. USA(1983) 80:726-730.

The “percent identity” of two amino acid sequences or of two nucleicacids is determined using the algorithm of Karlin and Altschul (Proc.Natl. Acad. Sci. USA 87:2264-2268, 1990), modified as in Karlin andAltschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such analgorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12. BLAST protein searches are performed with the XBLASTprogram, score=50, wordlength=3. Where gaps exist between two sequences,Gapped BLAST is utilized as described in Altschul et al. (Nucleic AcidsRes. 25:3389-3402, 1997). When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs(e.g.,)(BLAST and NBLAST) are used. See http://www.ncbi.nlm.nih.gov.

This invention also relates to a DNA encoding the above protein. Thereis no particular restriction as to the DNA of the present invention aslong as it encodes the protein of the present invention and includescDNA, genomic DNA and chemically synthesized DNA. cDNA can be preparedby, making a primer using the nucleotide sequence of the “SGRF” cDNA,disclosed in SEQ ID NO:2, and performing RT-PCR using the mRNA preparedfrom cells expressing the “SGRF” protein as the template. In the case ofgenomic DNA, preparation can be done by the plaque hybridization methodusing a genomic DNA inserted λ phage library and the cDNA probeobtained. The nucleotide sequence of the DNA acquired can be decided byordinary methods in the art using the commercially available “dyeterminator sequencing kit” (Applied Biosystems). The DNA of the presentinvention, as stated later, can be utilized for the production of arecombinant protein and gene therapy.

An “isolated nucleic acid” is a nucleic acid the structure of which isnot identical to that of any naturally occurring nucleic acid or to thatof any fragment of a naturally occurring genomic nucleic acid spanningmore than three separate genes. The term therefore covers, for example,(a) a DNA which has the sequence of part of a naturally occurringgenomic DNA molecule but is not flanked by both of the coding sequencesthat flank that part of the molecule in the genome of the organism inwhich it naturally occurs; (b) a nucleic acid incorporated into a vectoror into the genomic DNA of a prokaryote or eukaryote in a manner suchthat the resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.Specifically excluded from this definition are nucleic acids present inmixtures of different (i) DNA molecules, (ii) transfected cells, or(iii) cell clones: e.g., as these occur in a DNA library such as a cDNAor genomic DNA library.

The term “substantially pure” as used herein in reference to a givenpolypeptide means that the polypeptide is substantially free from otherbiological macromolecules. The substantially pure polypeptide is atleast 75% (e.g., at least 80, 85, 95, or 99%) pure by dry weight. Puritycan be measured by any appropriate standard method, for example, bycolumn chromatography, polyacrylamide gel electrophoresis, or HPLCanalysis.

The present invention also features a vector into which the DNA of thepresent invention has been inserted. There is no restriction as to thevector to which DNA is inserted, and various vectors such as those forexpressing the protein of the present invention in vivo and those forpreparing the recombinant protein can be used according to theobjective. To express the protein of the present invention in vivo(especially for gene therapy), various viral vectors and non-viralvectors can be used. Examples of viral vectors are, adenovirus vectors(pAdexLcw and such) and retrovirus vectors (pZlPneo and such).Expression vectors are especially useful when using vectors for theobjective of producing the protein of the invention. For example, whenusing Escherichia coli the “pQE vector” (Qiagen, Hilden, Germany), whenusing yeast “SP-Q01” (Stratagene, La Jolla, Calif.), when using insectcells “Bac-to-Bac baculovirus expression system” (GIBCO-BRL,Gaithersburg, Md.) are highly appropriate, but there is no restriction.Also, when using mammalian cells such as CHO cells, COS cells, NIH3T3cells, for example, the “LacSwitch II expression system (Stratagene, LaJolla, Calif.) is highly suitable, but there is no restriction.Insertion of the DNA of the present invention into a vector can be doneusing ordinary methods in the art.

The present invention also refers to a transformant, carrying, in anexpressible manner, the DNA of the present invention. The transformantof the present invention includes, those carrying the above-mentionedvector into which DNA of the present invention has been inserted, andthose host genomes into which the DNA of the present invention has beenintegrated. As long as the DNA of the present invention is maintained inan expressible manner, no distinction is made as to the form ofexistence of the transformants. There is no particular restriction as tothe cells into which the vector is inserted. When using the cells toexpress the protein of the present invention for the purpose of genetherapy by the ex vivo method, various cells (for example, various cellsof the immune system) can be used as target cells according to the typeof disease. Also, when the purpose is to produce the protein of thepresent invention, for example, E. coli, yeast, animal cells and insectcells can be used in combination with the vectors that are utilized.Introduction of a vector into a cell can be done using commonly knownmethods such as electroporation and calcium phosphate method.

The separation and purification of the recombinant protein from thetransformant made to produce the protein can be done using ordinarymethods. The recombinant protein can be purified and prepared by, forexample, ion exchange chromatography, reverse phase chromatography, gelfiltration, or affinity chromatography where an antibody against theprotein of the present invention has been immobilized in the column, orby combining two or more of these columns. Also when expressing theprotein of the present invention inside host cells (for example, animalcells and E. coli) as a fusion protein with glutathione-S-transferaseprotein or as a recombinant protein supplemented with multiplehistidines, the expressed recombinant protein can be purified using aglutathione column or nickel column. After purifying the fusion protein,it is also possible to exclude regions other than the objective proteinby cutting with thrombin or factor-Xa as required.

The present invention also features an antibody binding to the proteinof the invention. There is no particular restriction as to the form ofthe antibody of the present invention and include, apart from polyclonalantibodies, monoclonal antibodies as well. The antiserum obtained byimmunizing animals such as rabbits with the protein of the presentinvention, polyclonal and monoclonal antibodies of all classes,humanized antibodies made by genetic engineering, human antibodies, arealso included. The antibodies of the present invention can be preparedby the following methods. Polyclonal antibodies can be made by,obtaining the serum of small animals such as rabbits immunized with theprotein of the present invention, attaining a fraction recognizing onlythe protein of the present invention by an affinity column coupled withthe protein of the present invention, and purifying immunoglobulin G orM from this fraction by a protein G or protein A column. Monoclonalantibodies can be made by immunizing small animals such as mice with theprotein of the present invention, excising the spleen from the animal,homogenizing the organ into cells, fusing the cells with mouse bonemarrow cells using a reagent such as polyethylene glycol, selectingclones that produce antibodies against the protein of the invention fromthe fused cells (hybridomas), transplanting the obtained hybridomas intothe abdominal cavity of a mouse, and extracting ascites. The obtainedmonoclonal antibodies can be purified by, for example, ammonium sulfateprecipitation, protein A or protein G column, DEAE ion exchangechromatography, or an affinity column to which the protein of thepresent invention is coupled. The antibody of the invention can be usedfor purifying and detecting the protein of the invention. It can also beused as a pharmaceutical drug to control the function of the presentprotein. When using the antibody as a drug for humans, in the point ofimmunogenicity, the human antibodies or the humanized antibodies areeffective. The human antibodies or humanized antibodies can be preparedby methods commonly known to one skilled in the art. For example, humanantibodies can be prepared by, immunizing a mouse whose immune systemhas been changed to that of humans, using the protein of the invention.Also, humanized antibodies can be prepared by, for example, cloning theantibody gene from monoclonal antibody producing cells and using the CDRgraft method which transplants the antigen-recognition site of the geneinto an already known human antibody.

The present invention also relates to a method for screening a chemicalcompound that binds to the protein of the invention. The screeningmethod of the invention includes the steps of, (a) exposing a testsample to the protein of the invention, (b) detecting the bindingactivity between the test sample and the protein of the invention, and(c) selecting a compound having an activity to bind to the protein ofthe invention. Any test sample can be used without particularrestrictions. Examples are, synthetic low molecular weight compoundlibraries, purified proteins, expression products of gene libraries,synthetic peptide libraries, cell extracts, and culture supernatants.Selection of a compound that has an activity to bind to the protein ofthe invention can be done using methods commonly known to one skilled inthe art.

The screening of a protein which binds to the protein of the inventioncan be done by, for example, creating a cDNA library from tissues orcells (for example, testis, lymph nodes and thymus) predicted to expressa protein binding to the protein of the invention using a phage vector(λgt11 and Zap11), expressing this cDNA library on LB-agarose, fixingthe expressed proteins on the filter, biotin-labeling the protein of theinvention or purifying it as a fusion protein with GST protein, reactingthis with the above-described filter, and detecting plaques expressingthe binding proteins using streptavidin or anti-GST antibody (WestWestern Blotting method) (Skolnik E Y, Margolis B, Mohammadi M,Lowenstein E, Fischer R, Drepps A, Ullrich A, and Schlessinger J (1991)Cloning of PI3 kinase-associated p85 utilizing a novel method forexpression/cloning of target proteins for receptor tyrosine kinases.Cell 65:83-90).

The screening of a protein binding to the protein of the invention orthe gene of the protein, can also be done by following “the two hybridsystem” (“MATCHMAKER Two-hybrid System”, “Mammalian MATCHMAKERTwo-hybrid Assay Kit”, “MATCHMAKER One-Hybrid System” (Clontech),“HybriZAP Two-Hybrid Vector System” (Stratagene), Reference, “Dalton, S.and Treisman, R. (1992) Characterization of SAP-1, a protein recruitedby serum response factor to the c-fos serum response element. Cell68:597-612”). Namely, the protein of the invention is fused to the SRFbinding region or GAL4 binding region and expressed in yeast cells. AcDNA library, is prepared from cells predicted to express a proteinbinding to the protein of the invention so as to express the ligandfused to the VP16 or GaL4 transcriptional activation region. The cDNAlibrary is then introduced into the above yeast cells and the cDNAderived from the library is isolated from the positive clones detected(when a protein binding to the protein of the invention is expressed inyeast cells, the binding of the two activates a reporter gene makingpositive clones detectable). The isolated cDNA is expressed byintroducing it into E. coli to obtain a protein encoded by the cDNA.Furthermore, a protein binding to the protein of the invention can bescreened by, applying the culture supernatants or cell extracts of cellspredicted to express a protein binding to the protein of the inventiononto an affinity column in which the protein of the invention isimmobilized and purifying the protein that binds specifically to thecolumn.

Also, the method of screening molecules which bind when the immobilizedprotein of the invention is exposed to synthetic chemical compounds, ornatural substance banks, or a random phase peptide display library, orthe method of screening using high-throughput based on combinatorialchemistry techniques (Wrighton N C; Farrel F X; Chang R; Kashyap A K;Barbone F P; Mulcahy L S; Johnson D L; Barret R W; Jolliffe L K; Dower WJ. Small peptides as potent mimetics of the protein hormoneerythropoietin, Science (UNITED STATES) Jul. 26, 1996, 273 p 458-68,Verdine G L., The combinatorial chemistry of nature. Nature (ENGLAND)Nov. 7, 1996, 384 p 11-13, Hogan J C Jr., Directed combinatorialchemistry Nature (ENGLAND) Nov. 7, 1996, 384 p 17-9) to isolate lowmolecular weight compounds, proteins (or the genes) and peptides aremethods well known to one skilled in the art.

The present invention also relates to a method for screening a compoundable to promote or inhibit the activity of the protein of the invention.It was found that the protein of the invention has aproliferation-supporting activity for bone marrow cells in the presenceof the kit ligand. Therefore, using this activity as an indicator,screening of a compound able to promote or inhibit activity of theprotein of the invention can be performed. Namely, this screening can bedone using the method comprising the steps of: (a) exposing the proteinof the invention and the kit ligand to mammalian bone marrow cells underthe presence of a test compound; (b) detecting the proliferation of thebone marrow cells; and (c) selecting a compound which promotes orinhibits the proliferation of bone marrow cells when compared with theassay in the absence of a test sample (control).

There are no particular restrictions as to the test sample used.Examples are, libraries of synthetic low molecular compounds, purifiedproteins, expression products of gene libraries, synthetic peptidelibraries, cell extracts and culture supernatants. The compound isolatedby the above-described screening of a protein binding to the protein ofthe invention may also be used as a test compound.

The protein of the present invention and the kit ligand may berecombinant or natural proteins. Also, as long as the activity ismaintained, may be a partial peptide. The kit ligand may also be acommercially available one.

Lin negative, Sca-1 positive and c-kit positive bone marrow cells arepreferred for the screening. Bone marrow cells that are additionallyCD34 positive are preferred more.

The culture conditions and detection of proliferation of bone marrowcells can be done, for example, in conformance with Example 11.

As a result of the detection, compared with the proliferation of bonemarrow cells in the absence of the test compound (control), if theproliferation of bone marrow cells is suppressed with the addition of atest compound, then the test compound is judged to be a compound (orincludes the compound), which inhibits the activity of the protein ofthe invention. On the other hand, if the proliferation is promoted bythe addition of the test compound (or includes the compound), it isjudged to be a compound that promotes the activity of the protein of theinvention.

The protein of the present invention can be used as a reagent inresearch to control the proliferation of cells of the lymphoid andhematopoietic systems. The compound isolated by the above-mentionedscreening, can be used as an inhibitor or promoter of the protein in theinvention. Moreover, the protein of the invention or these compounds canalso be utilized as drugs for the prevention and therapy of diseasesassociated with defects in cell proliferation and lymphoid andhematopoietic systems.

When using the protein of the invention or a compound that controls theactivity of the protein as drugs, they can be formulated into a dosageform using commonly known pharmaceutical preparation methods. Forexample, according to the need, the drugs can be taken orally (assugar-coated tablets, capsules, elixirs and microcapsules) or non-orally(such as, percutaneously, intranasally, bronchially, intramuscularly andintravenously) in the form of injections of sterile solutions orsuspensions with water or any other pharmaceutically acceptable liquid.For example, the protein of the invention or compounds controlling theactivity of the protein can be mixed with physiologically acceptablecarriers, flavoring agents, excipients, vehicles, preservatives,stabilizers and binders, in a unit dose form required for generallyaccepted drug implementation. The amount of active ingredients in thesepreparations makes a suitable dosage within the indicated rangeacquirable.

Examples for additives which can be mixed to tablets and capsules are,binders such as gelatin, corn starch, tragacanth gum and arabic gum;excipients such as crystalline cellulose; swelling agents such ascornstarch, gelatin and alginic acid; lubricators such as magnesiumstearate; sweeteners such as sucrose, lactose or saccharin; flavoringagents such as peppermint, Gaultheria adenothrix oil and cherry. Whenthe unit dosage form is a capsule, a liquid carrier, such as oil, canalso be included in the above ingredients. Sterile composites forinjections can be formulated following normal drug implementations usingvehicles such as distilled water used for injections.

Physiological saline, glucose, and other isotonic liquids includingadjuvants, such as D-sorbitol, D-mannose, D-mannitol, and sodiumchloride, can be used as aqueous solutions for injections. These can beused in conjunction with suitable solubilizers, such as alcohol,specifically ethanol, polyalcohols such as propylene glycol and polyethylene glycol, non-ionic surfactants, such as Polysorbate 80 (TM) andHCO-50.

Sesame oil or soy-bean oil can be used as a oleaginous liquid and may beused in conjunction with benzyl benzoate or benzyl alcohol as asolubilizer; may be formulated with a buffer such as phosphate andsodium acetate; a pain-killer such as procaine hydrochloride; astabilizer such as benzyl alcohol, phenol, and an anti-oxidant. Theprepared injection is filled into a suitable ampule.

One skilled in the art can suitably select the dosage and method ofadministration according to the body-weight, age, and symptoms of apatient.

For example, although there are some differences according to thepatient, target organ, symptoms and method of administration, the doseis about 1 μg to about 100 mg per day for a normal adult (weight 60 kg)when the protein is given as an injection.

For a compound controlling the activity of the protein of the invention,although it can vary according to the symptoms, the dosage is about 0.1to about 100 mg per day, preferably about 0.1 to about 50 mg per day andmore preferably about 0.1 to about 20 mg per day, when administeredorally.

When administering non-orally in the form of an injection to a normaladult (weight 60 kg), although there are some differences according tothe patient, target organ, symptoms and method of administration, it isconvenient to intravenously inject a dose of about 0.01 mg to about 30mg per day, preferably about 0.1 to about 20 mg per day and morepreferably about 0.1 to about 10 mg per day. Also, in the case of otheranimals too, it is possible to administer an amount converted to 60 kgof body-weight.

This invention also features a DNA containing at least 15 nucleotides,which can specifically hybridize with DNA encoding the “SGRF” protein.The term “specifically hybridize” as used herein, indicates thatcross-hybridization does not occur significantly with DNA encoding otherproteins, in the above-mentioned hybridizing conditions, preferablyunder stringent hybridizing conditions.

Such DNA can be utilized to detect DNA encoding the “SGRF” protein, asan isolation probe, and also as a primer for amplification.Specifically, the primers in SEQ ID NOs:3 to 20 can be given asexamples. Such DNA can also be used as an oligo nucleotide or aribozyme.

An antisense oligonucleotide is preferably an antisense oligonucleotideagainst at least 15 continuous nucleotides in the nucleotide sequence ofSEQ ID NO:2. The above-mentioned antisense oligonucleotide, whichcontains an initiation codon in the above-mentioned at least 15continuous nucleotides, is even more preferred.

Derivatives or modified products of antisense oligonucleotides can beused as antisense oligonucleotides. Examples are, lower class alkylphosphate modifications such as methyl-phosphonate-type orethyl-phosphonate-type and phosphothioate or phosphoramidate.

The term “antisense oligonucleotides” as used herein means, not onlythose in which the nucleotides corresponding to those constituting aspecified region of a DNA or mRNA are complementary, but also thosehaving a mismatch of one or more nucleotides, as long as DNA or mRNA andan oligonucleotide can specifically hybridize with the nucleotidesequence of SEQ ID NO:2.

Such DNAs are indicated as those having, in the “at least 15 continuousnucleotide sequence region”, a homology of at least 70% or higher,preferably at 80% or higher, more preferably 90% or higher, even morepreferably 95% or higher to the nucleotide sequence of SEQ ID NO:2. Thealgorithm stated herein can be used to determine homology.

The antisense oligonucleotide derivative of the present invention, actsupon cells producing the protein of the invention by binding to the DNAor mRNA encoding the protein and inhibits its transcription ortranslation, promotes the degradation of the mRNA, inhibiting theexpression of the protein of the invention resulting in the inhibitionof the protein's function.

The antisense oligonucleotide derivative of the present invention can bemade into an external preparation such as a liniment and a poultice bymixing with a suitable base material, which is inactive against thederivatives.

Also, as needed, the derivatives can be formulated into tablets,powders, granules, capsules, liposome capsules, injections, solutions,nose-drops and freeze-drying agents by adding excipients, isotonicagents, dissolving auxiliaries, stabilizers, preservatives andpain-killers. These can be prepared using usual methods.

The antisense oligonucleotide derivative is given to the patient by,directly applying onto the ailing site or by injecting into a bloodvessel so that it will reach the site of ailment. An antisense-mountingmedium can also be used to increase durability andmembrane-permeability. Examples are, liposome, poly-L lysine, lipid,cholesterol, lipofectin or derivatives of these.

The dosage of the antisense oligonucleotide derivative of the presentinvention can be adjusted suitably according to the patient's conditionand used in desired amounts. For example, a dose range of 0.1 to 100mg/kg, preferably 0.1 to 50 mg/kg can be administered.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the “SGRF” cDNA nucleotide sequence together with thededuced amino acid sequence. The mRNA destabilizing sequence isunderlined.

FIG. 2 shows the alignment of the consensus sequence of “SGRF” andproteins belonging to the IL-6/G-CSF/MGF family. The consensus sequenceof this family is thought to be C-x(9)-C-x(6)-G-l-x(2)-[FY]-x(3)-L, iswell preserved in “SGRF” as well, excluding the point that the number ofthe first x is 11 instead of 9. In the figure, Sooty Mangabey is ananimal of the Cercocebus species and Rhesus Macaque is of the RhesusMonkey species.

FIG. 3 shows the hydrophobicity of “SGRF”.

FIG. 4 shows the electrophoretic pattern of the “SGRF” expression innormal human tissue as detected by Northern blot analysis. Markers are,from the left, 9.5 kb, 7.5 kb, 4.4 kb, 2.4 kb, and 1.35 kb.

FIG. 5 shows the electrophoretic pattern of the “SGRF” expression inhuman fetal tissue and tumor cells as detected by Northern blotanalysis. Markers are, from the left, 9.5 kb, 7.5 kb, 4.4 kb, 2.4 kb,and 1.35 kb.

FIG. 6 shows the amino acid sequence of a protein presumed to be thegenomic DNA nucleotide sequence of SGRF. Introns are shown in simpleletters, exons in capitals. Sequences expected to be those of the TATABox and poly A addition signal are underlined.

FIG. 7 shows the result of PCR analysis using NIGMS human/rodent somaticcell hybrid mapping panel #2. The numbers show the human chromosomescontained in the hybridoma DNA. ♀ shows human (female) genomic DNA, Mshows mouse genomic DNA. 100 bp ladder has been used as a marker. Theband seen around 200 bp is thought to be a non-specific backgroundderived from mouse chromosomes.

FIG. 8 shows the pCHO-SGRFg map.

FIG. 9 shows the pCHO-SGRF map.

FIG. 10 shows the pLG-SGRF map.

FIG. 11 shows the pLG-SGRFg map.

FIG. 12 shows the effect of the SGRF-gene-inserted, CHO cell culturesupernatant on the proliferation of bone marrow cells.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be explained in detail below with referenceto examples, but it is not limited thereto.

EXAMPLE 1 Isolation of the “SGRF” Gene

When the GenBank EST database was searched by means of TBLAST using thehuman G-CSF protein sequence, the EST of Reg. No: AA418955 showed a weakhomology to G-CSF. Based on this sequence, when an EST sequenceconsidered to be reading the same gene was searched, four otherregistered ESTs (AA418747, U38443, AA729815, AA418955) were found. Thesesequences were aligned using DNASIS, the consensus sequence wasextracted, and the following primers were designed:

“ILX-1” (GAGAAGAGGGAGATGAAGAGACTAC/SEQ. ID. NO: 3); “ILX-2”(CTGAGTCCTTGGGGGTCACAGCCAT/SEQ. ID. NO: 4); “ILX-3”(GTGGGACCTGCATATGTTGAAAATT/SEQ. ID. NO: 5; “ILX-4”(CCCCAAATTTCCCTTCCCATCTAATA/SEQ. ID. NO: 6); “ILX-5”(CCCTACTGGGCCTCAGCCAACTCCT/SEQ. ID. NO: 7);  and “ILX-6”(GGAGCAGAGAAGGCTCCCCTGTGAA/SEQ. ID. NO: 8.

Using the human fetal spleen library (Marathon-Ready cDNA; Clontech),sequential

PCR was performed in combinations of primers stated below, divided into3 fragments, and amplified separately (5′side, central area, and3′side). The primers used for the 5′side amplification were, “AP1”(Clontech) and “ILX-6” in the primary PCR, “AP2” (Clontech) and “ILX-2”in the nested PCR. As to the central area, “ILX-1” and “ILX-4” were usedfor the primary and nested PCRs. For the 3′ side, “AP1” (Clontech) and“ILX-5” were used in the primary PCR, “AP2” (Clontech) and “ILX-3” inthe nested PCR. Amplifications by both the primary and nested PCRs weredone under conditions in which those recommended by the Manufacturerwere partially changed (touchdown PCR: 1 min at 96° C., following 30 secat 96° C., 5 cycles of “4 min at 72° C”, following 30 sec at 96° C., 5cycles of “4 min at 70° C”, following 20 sec at 96° C. and 26 cycles of“4 min at 68° C”. However, TaKaRa Ex Taq (Takara Shuzo) and attachedbuffers were used instead of Advantage Klentaq Polymerase Mix). Theobtained DNA were then electrophoresed on agarose gel, the correspondingbands were cut-off, after purifying by QIAEX II Gel Extraction Kit(QIAGEN), were cloned into the plasmid pT7Blue (R) T-vector (Novagen).The obtained plasmids were named pT7Blue-ILX1-4 (the vector which clonedthe central area), pT7Blue-ILX5′ (the vector which cloned the 5′endarea), and pT7Blue-ILX3′ (the vector which cloned the 3′end area),respectively. Each nucleotide sequence cloned was determined using ABIPRISM Dye Terminator Cycle Sequencing Ready Reaction Kit with AmplitaqDNA Polymerase FS and 377 A DNA Sequencer (Perkin-elmer).

As a result, the full length of the isolated cDNA was 1 Kb, and encodeda protein comprising 189 amino acids (FIG. 1). This protein, not onlyhad a consensus sequence typical of IL-6/G-CSF/MGF family (FIG. 2), butalso a hydrophobic region considered to be a signal peptide in the Nterminal (FIG. 3). Also, the binding site of N-type sugar-chain was notseen. In the 3′ non-coding region, four mRNA destabilizing sequencescalled ARE (AT Rich element), often seen in cytokine mRNAs (FIG. 1),were detected, having characteristics of a typical cytokine. Based onthe structural homology, this molecule was named “SGRF”(Interleukin-Six, G-CSF Related Factor).

EXAMPLE 2 Northern Blot Analysis of “SGRF” Expression

A 500 bp fragment obtained by treating pT7Blue-ILX1-4 with BamHI wasused as the probe of “SGRF”. The above probe was “□-32P”dCTP labeled byrandom priming method using Ready-to Go DNA labeling beads (Pharmacia),and hybridization was done according to methods recommended by theManufacturer within the ExpressHyb Hybridization Solution (Clontech)against the Multiple Tissue Northern Blot (Human, Human III, Human IV,Human Fetal II, Human Cancer Cell Line (Clontech) filters. As a result,in normal tissues, “SGRF” was mainly expressed in the testis and lymphnodes, and an mRNA of approximately 1 kb was detected. An extremely lowexpression was found in the thymus. However, since a longautoradiography (1 week) was required for the detection of these bands,the expression level in these are considered to be low. “SGRF” mRNA wasnot in a detectable level in other tissues.

When cancer cell lines were analyzed, very strong levels of expressionwere detected in two cell lines; K562 (Chronic Myelogenous Leukemia) andSW480 (Colorectal Adenocarcinoma) (FIG. 5). In the other cell lines,“SGRF” mRNA was not in a detectable level.

EXAMPLE 3 Construction of the “SGRF” Expression Vector

Two primers, “ILXATG” TTGAATTCCACCATGCTGGGGAGCAGAGCTGT/SEQ ID NO:14),“ILXTAA” (AAAGATCTTAGGGACTCAGGGTTGCTGC/SEQ ID NO:15), were constructed,a gene consisting all the coding regions reconstituted by pT7Blue-ILX1-4and pT7Blue-ILX5′, and introduced into an animal cell expression vector.Namely, the band amplified using pT7Blue-ILX5′as the template and“ILX-2” and “ILXATG” as primers, and the band amplified usingpT7Blue-ILX1-4 as the template and “ILX-1” and “ILXTAA” as primers, weremixed in equal amounts, and re-amplification was done using this as thenew template with the primers “ILXATG” and “ILXTAA”. The resulting bandwas treated with restriction enzymes EcoRI and BamHI, and cloned intothe EcoRI, Bgl II site of an animal cell expression plasmid pCOS1 tocreate pCOS-SGRF. TaKaRa ExTaq (Takara Shuzo) was used for DNAamplification for 20 cycles of 30 sec at 96° C., following 40 sec at 60°C., and following 1 min 20 sec at 72° C.

EXAMPLE 4 The Polyclonal Antibody Corresponding to “SGRF”

Two kinds of partial peptides of “SGRF” (GGSSPAWTQCQQLSQ/24-38 positionof the amino acid sequence of SEQ ID NO:1, GDGCDPQGLRDNSQF, 74-88position of the amino acid sequence of SEQ ID NO:1) were chemicallysynthesized. Two rabbits were immunized with these, respectively, toobtain polyclonal antibodies (Sawady). The respective antibodies wereaffinity-purified using respective peptide columns. The followinganalyses were done using one of the antibodies against the peptide ofSGRF (24-38 position of the amino acid sequence of SEQ ID NO:1).

Alkyl phosphatase-binding, mouse-anti-rabbit IgG antibody and alkylphosphatase substrate were used for detection.

EXAMPLE 5 Genomic DNA of SGRF

The following sequences were prepared and used for the analysis of thepromoter regions-5′non translating region, translating region, 3′non-translating region from the genomic DNA library.

(SEQ ID NO: 3) ILX-1 5′-GAGAAGAGGGAGATGAAGAAGACTAC-3′ (SEQ ID NO: 4)ILX-2 5′-CTGAGTCCTTGGGGGTCACAGCCAT-3′ (SEQ ID NO: 5) ILX-35′-GTGGGACCTGCATATGTTGAAAATT-3′ (SEQ ID NO: 6) ILX-45′-CCCCAAATTTCCCTTCCCATCTAATA-3′ (SEQ ID NO: 7) ILX-55′-CCCTACTGGGCCTCAGCCAACTCCT-3′ (SEQ ID NO: 8) ILX-65′-GGAGCAGAGAAGGCTCCCCTGTGAA-3′ (SEQ ID NO: 9) ILX-75′-GGGCAGAGATTCCAGGAGGACTGGT-3′ (SEQ ID NO: 10) ILX-85′-CCAGTCCTGGTGGAATCTCTGCCCA-3′ (SEQ ID NO: 11) ILX-95′-GAAGCTCTGCACACTGGCCTGGAGT-3′ (SEQ ID NO: 12) ILX-105′-CACTCCAGGCCAGTGTGTGCAGAGCTT-3′ (SEQ ID NO: 13) ILX-115′-CTGAAGGGCTATGGTGGAGAA-3′ (SEQ ID NO: 14) ILX-ATG5′-TTGAATTCCACCATGCTGGGGAGCAGAGCTGT-3′ (SEQ ID NO: 15) ILX-TAA5′-AAAGATCTTAGGGACTCAGGGTTGCTGC-3′ (SEQ ID NO: 16) ILX-TAAECO 5′-AAGAATTGTAGGGACTCAGGGTTGCTGC-3′ (SEQ ID NO: 17) SGRFg5′5′-GGTTTAAATATTTGTTCTCCCTTACCCC-3′ (SEQ ID NO: 18) SGRFg375′-TTCAGCTGCTTGGGAGGCTGAGGCAGG-3′ (SEQ ID NO: 19) SGRFg5′-2 5′-AGGAATTCCACCAGGACTGGTGCAAGGCGCA-3′ (SEQ ID NO: 20) SGRFg3′-25′-GTCTCGAGAAAATATCATTCTCCACCATCGCCCT-3′

Genomic DNA was amplified by PCR using, for the translation region, theabove mentioned ILX-ATG primer and ILX-TAA primer and human genomic DNA(Clontech) as the template, resulting in a band amplified toapproximately 1.5 kb. After treating this fragment with restrictionenzymes EcoRI and BglII, cloning was done by inserting into theEcoRI-BamHI site of the CHO expression plasmid pCHO1. The nucleotidesequence of the vector obtained (pCHO-SGRFg) (FIG. 8) was analyzed usingthe primers described above. As a result, it was revealed to be the SGRFgene including 3 introns.

The amplifications of the 5′ non-translating region containing thepromoter, and the 3′ non-translating region, were done using GenomeWalker Kit as the template (Clontech), the attached AP1 and AP2 primersand the above-mentioned synthetic primers according to methodsrecommended by the Manufacturer.

First, for the 5′ non-translating region, the 1^(st) PCR was done usingDral library as the template with AP1 and ILX-10 primers. Then, the2^(nd) PCR was done with the AP2 and ILX-8 primers to obtain a band ofapproximately 400 bp.

For the 3′ non-translating region, the 1^(st) PCR was done using PvuIIlibrary as the template with AP1 and ILX-5 primers, the 2^(nd) PCR withthe AP2 and ILX-3 primers to obtain a band of app 800 bp.

The bands obtained were cut off from the agarose gel, and afterpurification, sequencing was done using 377 A DNA Sequencer(Perkin-Elmer). The genomic DNA sequence of SGRF (SEQ ID NO:21) is shownin FIG. 6 together with the deduced amino acids.

Also, NIGMS human/rodent somatic cell hybrid mapping panel #2 andGeneBridge 4 Radiation Hybrid Panel (Research Genetics) were analyzed byPCR using ILX-1 and Ilx-6 primers to examine the chromosome location. Asa result, from NIGMS human/rodent somatic cell hybrid mapping panel #2analysis, it was revealed that the SGRF gene exists on the 12^(th)chromosome (FIG. 7).

The analysis from GeneBridge 4 Radiation Hybrid Panel, revealed thatSGRF exists in 12q13 and is, Chromosome Chr12, Places 8.77 cR fromWI-7107 (lod>3.0).

EXAMPLE 6 Establishment of a CHO Cell Line Expressing SGRF

Similarly to pCOS-SGRF described in Example 3, DNA fragment of SGRFencoding region was prepared, was cloned to the EcoRI, BamHI site of theanimal cell expression plasmid pCHO1 to create pCHO-SGRF (FIG. 9).PCHO-SGRF was then transfected into CHO cells by calcium phosphatemethod and gene-introduced cells were selected in alpha-MEM culturemedium, which does not contain nucleotides. The culture supernatant wasanalyzed by SDS-PAGE and Western blotting using rabbit-polyclonalantibody.

As a result, a band with a molecular weight of about 20,000 was detectedonly in the culture supernatant of CHO cells having this vector.

Thereafter, the MTX concentration was increased sequentially to 20 nM,100 nM and so on, and the gene was amplified while verifying theexpression to establish a CHO cell strain, which constitutively secretesSGRF. This cell strain has been deposited in the depository institutiongiven below.

(a) Name and address of the Depository Institution

-   -   Name: National Institute of BioScience and Human-Technology        Agency of Industrial Science and Technology    -   Address: 1-3,Higashi-1-chome, Tsukuba-shi, Ibaraki 305-8566,        Japan.

(b) Date of deposit (date of original deposit): Apr. 9, 1999.

(c) Accession Number: FERM BP-6699

EXAMPLE 7 Purification of SGRF

In order to review purification of SGRF, producing cells (CHO-SGRF 16-5strain) proliferated to a confluent state, were rinsed with PBS, themedium was changed to a serum-free culture medium ASF 104 (AJINOMOTO),cultured for 3 to 4 days, and the culture supernatant was collectedafter filtering.

A 30 ml column was prepared using Phenyl-Sepharose HP (AmershamPharmacia Biotech), equilibrated by 10 mM Tris pH 7.5, 100 mM NaCl, andthe culture supernatant of the above mentioned CHO-SGRF16-5 straincultured in ASF medium, was 1.5 times diluted using 10 mM Tris pH 7.5and applied onto the column. After washing well with the equilibratingbuffer, extraction was done with the same buffer containing 0.1% Tween20. The extracted solution was applied to a DEAE-Sepharose FF columnequilibrated with 10 mM Tris pH 7.5, 100 mM NaCl, washed well with theequilibrating buffer, and extracted using 10 mM Tris pH 7.5, 300 mMNaCl, recovering most of the SGRF. The extracted sample was according tonormal methods by SDS-PAGE analysis, Western blot analysis as a crudelypurified product. As a result, a band binding to the polyclonal antibodywas detected at the site of a molecular weight of around 20,000 whichwas concentrated enough to be detected by Silver-staining andCoomassie-staining.

This crudely purified SRF protein was blotted onto a PVDF membrane,stained with Coomassie blue, and a band with a molecular weight of about20,000 was cut off to determine the N-terminal amino acid sequence usingModel 492 protein sequencer (Applied Biosystems). As a result, thesequence was found to be X-Ala-Val-Pro-Gly-Gly-Ser. This matched theSGRF sequence of 20th Arg from the N-terminal to the 29th Ser, and thesignal peptide was found to be cleaved between the 19th GLY and the 20thArg.

From the above results, the mature protein of SGRF was calculated to behaving 170 amino acids with a presumed molecular weight of 18,676 and anexpected isoelectric point of 5.84.

EXAMPLE 8 SGFR Vector for the Creation of a Transgenic Mouse

SGRF cDNA was amplified from pCHO-SGRF using the primers ILX-ATG andILX-TAAECO, cleaved with the restriction enzyme EcoRI, then insertedinto the EcoRI site of transgenic expression plasmid pLG1 to createpLG-SGRF (FIG. 10).

Also, the region containing SGRF genomic DNA was amplified from humangenomic DNA(clontech) using primers SGRF-5′_(—)2 and SGRF-3′_(—)2,treated with the restriction enzyme EcoRI, and then inserted into theEcoRI-XhoI site of the plasmid pLG1 to create pLG-SGRFg (FIG. 11).

EXAMPLE 9 Production of the Monoclonal Antibody

Five 8-week male Balb/c mice are immunized 2 mg/head with aluminumhydroxide gel as the adjuvant, and 20 μg/head of the above mentionedSGRF protein or the partial peptide as the antigen by injection into theperitoneal cavity. Re-immunization is done six times in every 2 weeks byinjecting 20 μg/head of the above mentioned SGRF protein or the partialpeptide. After the 3^(rd) immunization, blood is drawn from theeye-ground venous plexus and anti SGRF antibody titer in the serum isexamined.

Three days after the final immunization, spleen cells are prepared frommice, and used for cell fusion. 1×108 splenocytes from the immunizedmice washed well with MEM (Nissui Pharmaceuticals), and murine myelomaP3-U 1×108 are mixed and centrifuged for 5 min at 1000 rpm. 2 gPolyethylene glycol-1500 (PEG-1500), and 2 ml MEM are added while mixingwell at 37° C. and centrifuged after 1 min at 600 rpm for 5 min.Further, 5 ml HBSS solution and 5 ml 20% FBS/MEM solution are addedcalmly, cells are suspended well, and centrifuged at 1000 rpm after 1min, and the culture supernatant is discarded. The cells arere-suspended by adding 5 ml HAT medium (10−4M hypoxanthine, 4×10−7Maminopterin, and 1.5×10−5M thymidine supplemented medium). The cellsuspension is seeded in 1 ml/well into a 24-well culture plate (Nunc),and cultivated for 24 hr in a CO₂ incubator at 37° C., 5% CO₂, 95% air.1 ml/well HAT medium is added and cultured further for another 24 hr.Then, 1 ml of culture supernatant is discarded, 1 ml HAT medium is newlyadded and cultivated further for another 12 days.

For those wells in which colonized fusion cells can be detected, 1 mlculture supernatant is discarded, 1 ml HAT medium (aminopterin-excluded,above-mentioned HAT medium) added and cultured at 37° C. Exchange of theHAT medium is similarly done for the next two days and after culturingfor 4 days, a portion of the culture supernatant is collected to measurethe anti-SGRF antibody titer by ELISA.

For wells in which the antibody titer was detected, cloning is performedby limiting dilution twice more, and clones for which a stable antibodytiter was detected, are selected as hybridomas producing anti-SGRFmonoclonal antibody.

EXAMPLE 10 ELISA Method

50 μl/well of SGRF protein solution or the partial peptide solution isseeded into a 96-well culture plate (Immunoplate, Nunc) and is left tostand at room temperature for 2 hours to coat the antigen onto thebottom of the plate-well. Then, 200 μl/well of 10% FCS/PBS is added andleft to stand at room temperature for 30 min. The above-mentioned plateis washed 3 times with PBS, serially diluted test sample (mouse serum,hybridomas culture supernatant, monoclonal antibody and such) is seededin 50 μl/well, and left to stand at room temperature for 2 hours. Then,the plate is washed 3 times with PBS, and 100 times-dilutedperoxidase-binding goat-anti-mouse IgG antibody is seeded in 50 μl/wellas the secondary antigen and left to stand at room temperature for 2hours. After washing with PBS, 200 μl/well of peroxidase substrate (1%hydrogen peroxide, 0.1M acetic acid-0.05M phosphate buffer, 2 mM2,2′-azino-di-3-ethyl-benzothiazine sulfate) is added and after leavingat room temperature for 10 to 30 min, the antibody titer is calculatedusing calorimetry at 414 nm.

EXAMPLE 11

Bone marrow cells were prepared by extracting the thigh-bone andshank-bone of 8 to 15 week C57BL/6 male mice (CLEAR JAPAN). Aftersuspending in Nycodenz (Nycomed Pharm AS), the specific gravity wasadjusted to 1.063, stratified to NycoPrep 1.077 Animal (Nycomed PharmAS) and centrifuged for 30 min at 2300 rpm (Hitachi, 05PR22), 20° C. Theintermediate layer was collected, suspended in FACS buffer (2% fetalBovine Serum (FBS, Moregate) containing Dulbecco's phosphate buffer),was collected by centrifuging for 10 min at 1500 rpm, after which 1 μgof biotin-labeled anti-Mac-1 antibody, biotin-labeled anti-Gr-1antibody, biotin-labeled anti-TER119 antibody, biotin-labeled anti-CD3εantibody, and biotin-labeled B220 antibody (all by Pharmingen) wereadded per 1×106 cells. After leaving aside for 30 min on ice, was washedwith FACS buffer, 1 μl avidin-labeled microbeads (10 Beads Avidin,ImmunoTech) were added and left to stand for 15 min on ice. Beads werethen excluded by a magnetic holder, the floating cells were collected bycentrifuging for 1 min (Tomy MRX-150) at 5000 rpm. After discarding thesupernatant, 1 μg per 1×106 cells of FITC-labeled anti-CD34 antibody,PE-labeled anti Sca-1 antibody, APC-labeled c-kit antibody (all 3 byPharmingen), RED613-lebeled streptavidin (LifeTech Oriental) were addedand reacted for 30 min on ice. After washing with FACS buffer, wassuspended in 1 ml FACS buffer for 1×10⁶ cells, and fractionated by EPICSELITE (Beckman Coulter). The definitions of the respective fractions areas follows:

RED613-negative PE-negative APC-positive=Lin(−)Sca-1(−)c-kit(+)fraction

RED613-negative PE-positive APC-positive=Lin(−)Sca-1(+)c-kit(+)fraction

RED613-negative PE-positive APC-positiveFITC-positive=Lin(−)Sca-1(−)c-kit(+)CD34(+)fraction

RED613-negative PE-positive APC-positiveFITC-negative=Lin(−)Sca-1(+)c-kit(+)CD34(−)fraction

The obtained cell fractions were diluted by the Iscove's modifiedDulbecco's medium (10% FBS/IMDM) so that there were 10,000, 2000, and400 cells per 1 ml of medium and were seeded in 50 ml into a 96-wellculture plate.

To this, 50 μl of, (1) medium only (10% FBS/IMDM), (2) medium to whichmock has been diluted to 20% (mock), (3) SGRF-expressed CHO culturesupernatant (SGRF), (4) medium to which mouse kit ligand 10 ng/ml hasbeen added (KL), (5) medium to which 20% mock, 10 ng/ml mouse kit ligandhave been added (mock+KL), (6) SGRF-expressed CHO culture supernatant towhich 10 ng/ml mouse kit ligand has been added (SGRF+KL), (7) medium towhich 10 ng/ml mouse kit ligand, 5 ng/ml IL-11 has been added (KL+IL-11,were supplemented and then cultured for 10 days at 37° C., 5% CO₂.

Also, as for (1) and (3) mentioned above, 10 ng/ml, 1 ng/ml mouse kitligand IL-11 were added respectively to prepare a similarly cultured lotas well (medium/expand, mock/expand, SGRF/expand, respectively).

After completion of culture, cell number was detected using a Cellproliferation Assay Kit (Promega) as measured by Microplate Reader Model3550 (Bio-Rad) for the absorbance at 490 nm (FIG. 12).

As a result, although SGRF-alone showed no proliferation supportingactivity towards Lin negative, Sca-1 positive and c-kit positive cells,such a proliferation supporting activity was shown under the presence ofthe mouse kit ligand. This activity was stronger in CD34 positive cells.Also, if the mouse kit ligand did not exist at the initial stages ofculture, cells did not proliferate even when the ligand was supplementedafterwards. From this fact it can be assumed that SGRF does not have anactivity to support stem cells.

INDUSTRIAL APPLICABILITY

The present invention provided a novel cytokine-like protein and the DNAencoding the protein. Furthermore, a vector into which the DNA isinserted, a transformant possessing said DNA and the methods ofproduction of a recombinant protein are provided. A compound that bindsto the protein and a screening method for a compound that regulates itsactivity are also provided.

Since the protein of the invention and the gene, alike other cytokines,are believed to be associated with the activity or cell proliferationand differentiation of cells of the immune and hematopoietic systems,the use of a compound that controls said protein is anticipated indiseases relating to the immune or hematopoietic systems and defects incell proliferation. A number of embodiments of the invention have beendescribed. Nevertheless, it will be understood that variousmodifications may be made without departing from the spirit and scope ofthe invention.

1. A compound that promotes an activity of a polypeptide consisting ofthe sequence of SEQ ID NO:1.
 2. The compound of claim 1, wherein theactivity is supporting proliferation of bone marrow cells in thepresence of the kit ligand.
 3. A pharmaceutical composition comprisingthe compound of claim 1 as an active ingredient.
 4. The compound ofclaim 1, wherein the compound is obtained by a method comprising:contacting a mammalian bone marrow cell with a polypeptide comprisingthe sequence of SEQ ID NO:1 and a kit ligand, in the presence of a testcompound; detecting proliferation of the bone marrow cell; and selectingthe test compound if it promotes the proliferation of the bone marrowcell in comparison with a bone marrow cell cultured in the absence ofthe test compound.
 5. The compound of claim 4, wherein the bone marrowcell is Lin negative, Sca-1 positive, c-kit positive, and CD34 positive.6. A compound that inhibits an activity of a polypeptide consisting ofthe sequence of SEQ ID NO:1.
 7. The compound of claim 6, wherein theactivity is supporting proliferation of bone marrow cells in thepresence of the kit ligand.
 8. A pharmaceutical composition comprisingthe compound of claim 6 as an active ingredient.
 9. The compound ofclaim 6, wherein the compound is obtained by a method comprising:contacting a mammalian bone marrow cell with a polypeptide comprisingthe sequence of SEQ ID NO:1 and a kit ligand, in the presence of a testcompound; detecting proliferation of the bone marrow cell; and selectingthe test compound if it inhibits the proliferation of the bone marrowcell in comparison with a bone marrow cell cultured in the absence ofthe test compound.
 10. The compound of claim 9, wherein the bone marrowcell is Lin negative, Sca-1 positive, c-kit positive, and CD34 positive.11. A compound that specifically binds to a polypeptide consisting ofthe sequence of SEQ ID NO:1.
 12. A pharmaceutical composition comprisingthe compound of claim 11 as an active ingredient.
 13. The compound ofclaim 11, wherein the compound is obtained by a method comprising:contacting a test compound with a polypeptide comprising the sequence ofSEQ ID NO:1; determining whether the test compound binds to thepolypeptide; and selecting the test compound if it binds to thepolypeptide.